- Email: [email protected]
CYP2E1 activity in patients with alcoholic liver disease
Journal of Hepatology
1991; 27: 1009-1014 rights reserved Copenhagen
Printed in Denmark Munksgaard
Copyright 0 European Association for the Studv of the Liver 1997
. AN
Journal of Hepatology ISSN 0168-8278
CYP2El activity in patients with alcoholic liver disease Karin Dilger’, J&g Metzler*, Johann Christian
Bode* and Ulrich Klotz’
‘Dr. Margarete Fischer-Bosch-Institut fiir Klinische Pharmakologie, and 2Robert Bosch Krankenhaus, Stuttgart, Germany
Background/Aims: In addition to the possible toxicological impact of cytochrome P4502El (CYPZEl) in alcohol-induced liver damage, its activity can be regarded as a variable for drug action in patients with alcoholic liver disease as CYP2El is involved in the metabolism of several drugs, for example, paracetamol and halogenated anesthetics. The purpose of our study was to acquire detailed knowledge of CYP2El activity in patients with progressingly severe manifestations of alcoholic liver disease. Methods: The concentration ratio of 6-hydroxy-chlorzoxazone/chlorzoxazone in plasma 2 h after ingestion of 500 mg chlorzoxazone (so-called metabolic ratio) has been shown to reflect CYP2El activity in viva. We examined CYP2El activity in 56 Caucasian inpatients with minor (n=20), more pronounced (n=14) and severe alcoholic liver disease (n=22). Alcohol abusers were compared to healthy teetotallers (n= 14). Results: Metabolic ratios were increased 3-fold in actively drinking (ethanol-induced) compared to ab-
staining (non-induced) patients with alcoholic liver disease (1.19*0.84 vs. 0.44*0.45, mean&SD, (p
F
radical) metabolites than the parent compounds (7). Besides the possible toxicological implication of CYP2El in alcohol-induced tissue damage, CYP2El acitivity is important for drug action in patients with alcoholic liver disease as it is involved in the metabolism of several drugs, for example, paracetamol and halogenated anesthetics (8). Severe liver disease is commonly associated with impaired P450-mediated drug elimination, but there have also been reports of unchanged or increased rates of elimination of drugs known to be substrates for P450 enzymes (9). Chlorzoxazone, a centrally acting muscle relaxant and substrate for CYPZEl, is used as an in vivo probe for evaluating CYP2El activity in man (10-13). So far, there are only conflicting in vitro results (1416), but no clinical data are available concerning the question whether CYP2El activity is impaired in patients with severe alcoholic liver disease. The purpose of our study was therefore to acquire detailed knowledge of CYP2El activity by the chlorzoxazone test in patients
LEER, alcoholic hepatitis and alcoholic cirrhosis are progressively severe manifestations of alcoholic liver disease (ALD). The finding that only a minor proportion of alcoholics develops cirrhosis of the liver (1) suggests a genetic predisposition (2). Ethanol-inducible cytochrome P4502El (CYP2El) has been linked to individual susceptibility to the hepatotoxic effect of alcohol (3). However, the results of previous studies associating genetic polymorphism at the CYP2El locus and susceptibility to ALD are contradictory (4,5). CYP2El represents the key enzyme in the non-ADH oxidation of ethanol producing acetaldehyde, an established cytotoxin (6). CYP2El-mediated metabolism often generates more toxic (reactive free ATTY
Received 1 April; revised I7 June; accepted 14 July 1997
Correspondence: Ulrich Klotz, Dr. Margarete FischerBosch-Institut ftir Klinische Pharmakologie, Auerbachstr. 112, D-70376 Stuttgart, Germany. Tel: 071 l-81013702. Fax: 071 l-859295.
Key words: Alcoholic liver disease; Chlorzoxazone; Cytochrome P450.
1009
et al.
K. Dilger
with progressingly liver disease.
severe manifestations
duced; four women and 16 men), 14 patients with more pronounced ALD (three induced, eight non-induced, three tested twice induced and later non-induced; six women and eight men) and 22 patients with severe ALD (two induced, 20 non-induced; three women and 19 men). The diagnosis of ‘severe ALD’ (alcoholic liver cirrhosis) was based on clinical signs and symptoms (esophageal varices, ascites, splenomegaly), ultrasonic evidence of cirrhosis and deranged liver function tests (albumin, prothrombin time, bilirubin). Each patient with severe ALD was graded according to the Child classification (17) into class A (n=2), class B (n= 12) or class C (n=8). ‘More pronounced ALD’ (probably alcoholic hepatitis) was diagnosed if there were no clinical or ultrasonic signs of liver cirrhosis but elevated serum alanine aminotransferase and bilirubin levels (AST>SO U/l and total bilirubin > 1.4 mgidl). In seven patients, the diagnosis of alcoholic hepatitis was confirmed by liver biopsy. Alcohol abusers without clinical or ultrasonic evidence of liver cirrhosis, but with typical ultrasonic findings of fatty liver and AST and total bilirubin below the above limits, were assigned to the group of patients with ‘minor ALD’ (probably only fatty liver). Fourteen age-matched healthy Caucasians (five women and nine men) participated as controls. Health status was examined on the basis of history, physical examination and laboratory findings. They represented teetotallers (serum ethanol not detectable) and they did not take any medications. None was obese. Two subjects were smokers. Clinical and laboratory data of patients and healthy controls are given in Table 1.
of alcoholic
Materials and Methods Subjects All patients and control subjects were tested for chlorzoxazone metabolism, after informed consent was obtained from each individual, using a protocol that was approved by the ethics committee of our hospital according to the ethical guidelines of the 1975 Declaration of Helsinki. Fifty-six Caucasian inpatients with the diagnosis of alcoholic liver disease were included in the study. Alcohol consumption was evaluated by a face-to-face-interview concerning daily ethanol consumption, duration of alcohol abuse and time of last drink. Men (women) who had been drinking more than 60 g (20 g) of ethanol daily for at least 5 years were defined as ‘alcohol abusers’. Alcohol abusers were considered as ‘induced’ if their last drink was within the 48 h preceding the chlorzoxazone intake. Alcohol abusers were considered as ‘non-induced’ after at least 6 days of alcohol withdrawal, with serum ethanol concentrations below 0.1 g/l (limit of detection of TDx/ TDxFLx REA@ ethanol assay, Abbott, Wiesbaden, Germany). Patients with pancreatitis, HIV-infection, a positive hepatitis B surface antigen or a positive antiHCV (second-generation antibody) test were excluded from the study. Patients did not receive any medication that might interfere with CYP2El activity (paracetamol, disulfiram, isoniazid, chloramphenicol, halogenated inhalational anesthetics, clomethiazole). Diabetes, serious obesity and smoking might alter CYP2El activity and were therefore recorded: four patients were non-insulin-dependent diabetics, four had a body mass index greater than 30 kg/m2 and 27 were smokers. Blood samples were obtained from 20 alcohol abusers with minor ALD (three induced, 13 non-induced, four tested twice induced and later non-in-
TABLE
Chlovzoxazone test Early in the morning after a 12-h fast, each subject received a 500 mg tablet of chlorzoxazone (Parafon forte DSC@, McNeil Pharmaceutical, Spring House, NJ, USA) orally with sips of water. A venous blood
1
Clinical and laboratory data severe alcoholic liver disease
(normal
ranges
in parentheses)
Healthy n=14 Consumption
of ethanol,
Age, yr BMI, kg/m’ (obesity >30) AST, U/l (2-19) GGT, U/l (d 6-18, P 4-18) Albumin, g/d1 (3.5-5) Bilirubin, mgidl (0.2-l .4) PT, % (70-100)
controls
g/day 58.92 11.8 25.422.9 10.92 1.6 13.425.7 4.220.3 0.7-co.3 99.122.3
of healthy
controls
Minor n=17
ALD
131-c89 51.9t11.1 23.324.0 24.52 12.2** 164?210** 3.920.8 l.OiO.8 94.429.7
and non-induced
patients
More pronounced n=ll
ALD
148~ 124 49.528.5 25.123.2 107-t52*** 1345t1474*** 3.820.4 3.7t5.7** 89.5% 13.9
Data are means?SD. Abbreviations: ALD, alcoholic liver disease; BMI, body mass index; AST, alanine transferase; PT. prothrombin time; significances (patient groups vs. healthy): **p
1010
with minor,
more pronounced
and
Severe ALD n=20 111286 59.627.6 25.2~4.2 32t28*** 200-+353*** 3.1 t0.6*** 3.7-+4.5*** 57.8%10.3***
aminotransferase;
GGT.
y-glutamyl-
CYP2El and alcoholic liver disease
sample was withdrawn 2 h after drug intake to measure chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. Thereafter fasting was stopped. Serum was stored at -20°C until analysis. Analytical
methods
Chlorzoxazone and 6-hydroxy-chlorzoxazone were determined by HPLC as described previously (18). In brief, 0.5 ml of serum was added to 0.5 ml of 0.1 M sodium acetate buffer (pH 4.0) and 10 ,ul internal standard solution (pentoxifylline 1 mg/ml) and hydrolysed overnight at 37°C with 30 ,nl of P-glucuronidase solution from Helix pomatia @-Glucuronidase/Arylsulfatase@, Boehringer Mannheim, Mannheim, Germany). Proteins were then precipitated with 0.1 ml 70% perchloric acid. Chlorzoxazone and 6-hydroxy-chlorzoxazone were extracted with 5 ml dichloromethane. The organic phases were evaporated to dryness under a stream of nitrogen. The residues were dissolved in 250 ~1 of mobile phase and 30 ,~l were injected onto a 125X4 mm RP18-column (Nucleosil 100, 5 pm, purchased from Macherey-Nagel, Dtiren, Germany). The mobile phase consisted of 8% acetonitril, 10% phosphoric acid (5%) and 82% water (v/v/v). The separated compounds were detected at 287 nm. Retention times were 8.8 min for 6-hydroxy-chlorzoxazone, 29.8 min for chlorzoxazone and 55.5 min for the internal standard. Peak height ratios were used for quantification. Calibration curves ranged from 0.5 to 20 ,@ml and from 0.5 to 4 ,&ml for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. The 6-hydroxy-chlorzoxazone/chlorzoxazone concentration ratio was calculated and defined as metabolic ratio reflecting CYP2El activity Statistical
methods
The non-parametric Mann-Whitney test was used to assess the significance of differences between values for induced and non-induced alcohol abusers. By means of non-parametric ANOVA (Kruskal-Wallis, Dunn’s multiple comparisons test and post-test for linear trend, Graphpad Instat@), metabolic ratios and clinical and laboratory data were compared between three groups of non-induced patients and healthy controls. The effect of diabetes, obesity, smoking and sex on 6hydroxylation of chlorzoxazone was tested by multifactor ANOVA (Statgraphics Plus’s’). Correlation coefficients were derived by Spearman rank correlation (Graphpad Instat@).
Results No statistically significant differences between controls and patient groups were noted in age and body mass
healthy
minor
controls
ALD
more pronounced ALD
severe ALD
Fig. 1. Individual metabolic ratios in healthy controls (n= 14) and non-induced patients with minor (n=17), more pronounced (n=ll) and severe (n=20) alcoholic liver disease (ALD). Values from each group are given as means?SD. **Significantly d@erent (p
index. Consumption of ethanol (g/day) did not differ significantly between patients with minor, more pronounced and severe ALD. Metabolic ratios were measured in 15 ethanol-induced patients (seven minor, six more pronounced, two severe ALD) and 41 other noninduced patients with ALD (13 minor, eight more pronounced, 20 severe ALD). Seven (four minor, three more pronounced ALD) of the 15 induced patients were tested again after at least 6 days of ethanol withdrawal. Metabolic ratios were significantly greater in induced patients than in non-induced patients (1.1920.84, n=15 vs. 0.4320.45, n=48, mean+SD, p
olism between diabetic/non-diabetic, obese/non-obese, smoking/non-smoking and female/male subjects. In non-induced patients with severe ALD, there was no correlation between metabolic ratio and serum bilirubin (r=-0.22, p>O.O5), serum albumin (Y= -0.04, p>O.O5), prothrombin time (r=0.37, ~~0.05) and Child score (Y= -0.08, p>O.O5).
Discussion Ethanol intake induced metabolism of chlorzoxazone in alcohol abusers, Elevated CYP2El activity declined rapidly during withdrawal of alcohol. Previous studies have shown a 3-fold enhancement of the mean metabolic ratio in alcoholics tested within 2 days following ethanol withdrawal compared to alcoholics tested after 1 week of ethanol abstinence (18,19). Thus, our data are in very good agreement with these studies. Induction of CYP2El was also found in chemically-induced diabetic rats and in seriously obese humans (20-22). However, CYP2El activity was not altered in our few diabetic and obese patients. All diabetics were non-insulin-dependent and not ketoacidotic. Smoking status or sex did not appear to be of great importance when determining CYP2El activity in healthy and alcoholic subjects (18,19,23). Some investigators question the usefulness of chlorzoxazone as an in vivo probe for CYP2El activity because it has been suggested that both CYP2El and CYPlA1/2 are involved in the metabolism of chlorzoxazone. However, the contribution of CYPlA enzymes to chlorzoxazone 6-hydroxylation was considered negligible in several in vivo studies (24-28). In a recent investigation, neither human hepatic ADH nor human erythrocyte catalase contributed to chlorzoxazone metabolism, confirming the usefulness of chlorzoxazone in alcohol research on CYP2El activity (29). CYP2El activity declined continuously with severity of alcoholic liver damage in non-induced patients with ALD. We found the lowest metabolic ratios in patients with severe ALD (alcoholic liver cirrhosis). Although liver disease is well known to affect drug metabolism, the extent to which specific cytochrome P450 enzymes are impaired may vary strongly. The microsomal content of different cytochrome P450 enzymes was selectively altered in severe chronic liver disease, some being profoundly decreased, others less or not at all (14). CYP2El was reduced in microsomes of cirrhotic livers from patients with cholestasis, but not in other noncholestatic cirrhotic livers (14). In another study CYP2El levels were lower in liver samples from patients with cirrhosis than in normal samples or samples from patients with metastatic cancer (15). However, other patients with severe end-stage liver disease had 1012
normal levels of CYP2El protein (16). In viva activity of CYP2El has not yet been studied in patients with liver cirrhosis, whereas in vivo activities of several other hepatic drug metabolizing P450 enzymes, e.g. CYPlA2 and CYP2C19, were found to be reduced in these patients (30,31). It has been recommended that dosages of drugs that are substrates for disease-impaired CYPisoenzymes should be decreased in patients with liver dysfunction (9). Knowledge of altered CYP2El activity may permit better understanding or even prevention of certain adverse drug reactions, as CYP2EI is responsible for bioactivation of halothane, enflurane, isoflurane and paracetamol(32). There are consistent data indicating that both fulminant halothane hepatitis and paracetamol hepatotoxicity are associated with CYP2E 1-mediated bioactivation (33,34). Induction of CYP2E1, e.g. by ethanol, will aggravate drug-induced hepatotoxicity, whereas inhibition of CYP2EI might play a protective role. It is not known whether CYP2El is involved in fatal hepatocellular toxicity of chlorzoxazone (35). It is still an open question why some alcoholics develop severe ALD, whereas others consuming the same amount of alcohol appear to tolerate the hepatotoxic effects of ethanol. Cirrhosis morbidity starts to increase exponentially when daily alcohol consumption exceeds 60 g for male drinkers or 20 g for female drinkers (36). We studied only patients with ALD consuming ethanol beyond this critical threshold. As CYP2EI is involved in the generation of reactive free radical metabolites, oxidative species, acetaldehyde, and increased lipid peroxidation variable activity of this enzyme might explain interindividual susceptibility to ALD. Different restriction fragment length polymorphisms within the CYP2El gene have been suggested as genetic markers of susceptibility to ALD in heavy drinkers. Previous studies disagree whether alcoholics with the cl or c2 allele are at greater risk of ALD (4,5,3743). It is noteworthy that there is no relationship between genotype and phenotype of CYP2E 1, as differences in CYP2E 1 activity measured by 6-hydroxylation of chlorzoxazone in vitro and in vivo are not related to allelic forms of the CYP2El gene (25,38). Distinct phenotypes of CYP2E1, like slow and fast metabolizers. have not been described in the general population. In summary, we confirmed a 3-fold acceleration of CYP2El-mediated drug metabolism in drinking compared to abstaining patients with ALD. However, the major finding of the study was continuously decreasing CYP2El activity in abstaining patients with progressiveiy severe manifestations of ALD. It is not clear, whether alcoholic liver cirrhosis leads to reduced
CYP2El
CYP2El activity and thus impaired metabolism of chlorzoxazone or whether severe alcoholic liver injury is a consequence of altered CYP2El activity. Future investigations are necessary to determine whether CYP2El-mediated drug metabolism is reduced in all patients with advanced liver disease regardless of etiology. Nevertheless, it appears to be prudent to reduce the dosage of drugs which are substrates of CYP2El in patients with severe liver disease.
Acknowledgements 6-Hydroxy-chlorzoxazone was a kind gift of Dr. R. Boecker (University of Erlangen, Germany). This study was supported by the Robert Bosch Foundation, Stuttgart, Germany.
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and alcoholic liver disease
14. George J, Murray M, Byth K, Farrell G. Differential alterations of cytochrome P450 proteins in livers from patients with severe chronic liver disease. Hepatology 1995; 21: 1208. 15. Guengerich FP, Turvy CG. Comparison of levels of several human microsomal cytochrome P-450 enzymes und epoxide hydrolase in normal and disease states using immunochemical analysis of surgical liver samples. J Pharmacol Exp Ther 1991; 256: 1189-94. 16. Lown K, Kolars J, Turgeon K, Merion R, Wrighton SA, Watkins PB. The erythromycin breath test selectively measures P450IIIA in patients with severe liver disease. Clin Pharmacol Ther 1992; 51: 229-38. 17. Pugh RNH, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 60: 6469. 18. Girre C, Lucas D, Hispard E, Menez C, Dally S, Menez JE Assessment of cytochrome P4502El induction in alcoholic patients by chlorzoxazone pharmacokinetics. Biochem Pharmacol 1994; 47: 1503-8. 19. Lucas D, Menez C, Girre C, Bodenez P, Hispard E, Menez JE Decrease in cytochrome P4502El as assessed by the rate of chlorzoxazone hydroxylation in alcoholics during withdrawal phase. Alcohol Clin Exp Res 1995; 19: 362-6. 20. Song BJ, Matsunaga T, Hartwick JP, Park SS, Veech RL, Yang CS, et al. Stabilization of cytochrome P45Oj messenger ribonucleic acid in the diabetic rat. Mol Endocrinol 1987; 1: 542-7. 21. Dong Z, Hong J, Ma Q. Mechanism of induction of cytochrome P-450ac (P-450j) in chemically induced and spontaneously diabetic rats. Arch Biochem Biophys 1988; 263: 29-35. 22. O’Shea D, Davis SN, Kim RB, Wilkinson GR. Effect of fasting and obesity in humans on the 6-hydroxylation of chlorzoxazone: a putative probe of CYP2El activity. Clin Pharmaco1 Ther 1994; 56: 359-67. 23. Lucas D, Menez C, Girre C, Berthou F, Bodenez P, Joannet I, et al. Cytochrome P450 2El genotype and chlorzoxazone metabolism in healthy and alcoholic Caucasian subjects. Pharmacogenetics 1995; 5: 298-304. 24. Carriere V, Goasduff T, Ratanasavanh D, Morel F, Gautier JC, Guillouzo A, et al. Both cytochromes P450 2El and 1Al are involved in the metabolism of chlorzoxazone. Chem Res Toxic01 1993; 6: 852-7. 25. Yamazaki H, Guo Z, Guengerich FP Selectivity of cytochrome P4502El in chlorzoxazone 6-hydroxylation. Drug Metab Dispos 1995; 23: 438-9. 26. Berthou F, Goasduff T, Lucas D, Dreano Y, Le Bot MH, Menez JE Interaction between two probes used for phenotyping cytochromes P4501A2 (caffeine) and P4502El (chlorzoxazone) in humans. Pharmacogenetics 1995; 5: 72-9. 37 . Ono S, Hatanaka T, Hotta H, Tsutsui M, Satoh T, Gonzalez _, FJ. Chlorzoxazone is metabolized by human CYPlA2 as well as by human CYP2El. Pharmacogenetics 1995; 5: 143-50. 28. Carriere V, Berthou F, Baird S, Belloc C, Beaune P de Waziers I. Human cytochrome P450 2El (CYP2El): from genotype to phenotype. Pharmacogenetics 1996; 6: 203-l 1. 29. McCarver-May DG, Durisin L. Human hepatic alcohol dehydrogenase and human erythrocyte catalase do not metabolize the cytochrome P-4502El substrate, chlorzoxazone. Alcohol Clin Exp Res 1996; 20: 533-7. 30. Denaro CP Wilson M, Jacob III P, Benowitz NL. The effect of liver disease on urine caffeine metabolite ratios. Clin Pharmacol Ther 1996; 59: 624-35.
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K. Dilger et al. 3 1. Rost KL, Brockmiiller J, Esdorn F Roots I. Phenocopies of poor metabolizers of omeprazole caused by liver disease and drug treatment. J Hepatol 1995; 23: 268-77. 32. Leeder JS, Okey AB. Cytochromes P450 and liver injury. In: Cameron RG, Feuer G, Delaiglesia FA, editors. Drug-Induced Hepatoxicity. Berlin: Springer-Verlag; 1996. p. 11953. 33. Kharasch ED, Hankins D, Mautz D, Thummel KE. Identification of the enzyme responsible for oxidative halothane metabolism: implications for prevention of halothane hepatitis. Lancet 1996; 347: 1367. 34. Lee SST, Buters JTM, Pineau T, Fernandez-Salguero P, Gonzalez FJ. Role of CYP2El in the hepatotoxicity of acetaminophen. J Biol Chem 1996; 271: 1206337. 35. Nightingale SL. From the Food and Drug Administration: chlorzoxazone warning on hepatotoxicity is strengthened. JAMA 1995; 24: 1903. 36. Pequinot G, Tuyns AJ, Berta JL. Ascitic cirrhosis in relation to alcohol consumption. Int J Epidemiol 1978; 7: 113-20. 37. Stephens EA, Taylor JA, Kaplan N, Yang CH, Hsieh LL, Lucier GW, et al. Ethnic variation in the CYP2El gene: polymorphism analysis of 695 African-Americans, EuropeanAmericans and Taiwanese. Pharmacogenetics 1994; 4: 185% 92.
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38. Kim RB, Yamazaki H, Chiba K, O’Shea D, Mimura M, Guengerich FP et al. In vivo and in vitro characterization of CYP2El activity in Japanese and Caucasians. J Pharmacol Exp Ther 1996; 279: 4-11. 39. Carr LG, Hartleroad, JY, Liang Y, Mendenhall C, Moritz T, Thomasson H. Polymorphism at the P450IIEl locus is not associated with alcoholic liver disease in Caucasian men. Alcohol Clin Exp Res 1995; 19: 1824. 40. Lucas D, Menez C. Floch F, Gourlaouen Y, Sparfel 0, Joannet I, et al. Cytochromes P4502El and P4501Al genotypes and susceptibility to cirrhosis or upper aerodigestive tract cancer in alcoholic Caucasians. Alcohol Clin Exp Res 1996; 20: 1033-7. 41. Savoleinen VT, Pajarinen J, Perola M, Penttila A, Karhunen PJ. Polymorphism in the cytochrome P450 2El gene and the risk of alcoholic liver disease. J Hepatol 1997; 26: 55-61. 42. Agundez J, Ladero J, Diaz-Rubio M, Benitez J. Rsa I polymorphism at the cytochrome P4502El locus is not related to the risk of alcohol-related severe liver disease. Liver 1996; 16: 380-3. 43. Pirmohamed M, Kitteringham NR, Quest LJ, Allott RL, Green VJ, Gilmore IT, et al. Genetic polymorphism of cytochrome P4502El and risk of alcoholic liver disease in Caucasians. Pharmacogenetics 1995: 5: 351-7.
1991; 27: 1009-1014 rights reserved Copenhagen
Printed in Denmark Munksgaard
Copyright 0 European Association for the Studv of the Liver 1997
. AN
Journal of Hepatology ISSN 0168-8278
CYP2El activity in patients with alcoholic liver disease Karin Dilger’, J&g Metzler*, Johann Christian
Bode* and Ulrich Klotz’
‘Dr. Margarete Fischer-Bosch-Institut fiir Klinische Pharmakologie, and 2Robert Bosch Krankenhaus, Stuttgart, Germany
Background/Aims: In addition to the possible toxicological impact of cytochrome P4502El (CYPZEl) in alcohol-induced liver damage, its activity can be regarded as a variable for drug action in patients with alcoholic liver disease as CYP2El is involved in the metabolism of several drugs, for example, paracetamol and halogenated anesthetics. The purpose of our study was to acquire detailed knowledge of CYP2El activity in patients with progressingly severe manifestations of alcoholic liver disease. Methods: The concentration ratio of 6-hydroxy-chlorzoxazone/chlorzoxazone in plasma 2 h after ingestion of 500 mg chlorzoxazone (so-called metabolic ratio) has been shown to reflect CYP2El activity in viva. We examined CYP2El activity in 56 Caucasian inpatients with minor (n=20), more pronounced (n=14) and severe alcoholic liver disease (n=22). Alcohol abusers were compared to healthy teetotallers (n= 14). Results: Metabolic ratios were increased 3-fold in actively drinking (ethanol-induced) compared to ab-
staining (non-induced) patients with alcoholic liver disease (1.19*0.84 vs. 0.44*0.45, mean&SD, (p
F
radical) metabolites than the parent compounds (7). Besides the possible toxicological implication of CYP2El in alcohol-induced tissue damage, CYP2El acitivity is important for drug action in patients with alcoholic liver disease as it is involved in the metabolism of several drugs, for example, paracetamol and halogenated anesthetics (8). Severe liver disease is commonly associated with impaired P450-mediated drug elimination, but there have also been reports of unchanged or increased rates of elimination of drugs known to be substrates for P450 enzymes (9). Chlorzoxazone, a centrally acting muscle relaxant and substrate for CYPZEl, is used as an in vivo probe for evaluating CYP2El activity in man (10-13). So far, there are only conflicting in vitro results (1416), but no clinical data are available concerning the question whether CYP2El activity is impaired in patients with severe alcoholic liver disease. The purpose of our study was therefore to acquire detailed knowledge of CYP2El activity by the chlorzoxazone test in patients
LEER, alcoholic hepatitis and alcoholic cirrhosis are progressively severe manifestations of alcoholic liver disease (ALD). The finding that only a minor proportion of alcoholics develops cirrhosis of the liver (1) suggests a genetic predisposition (2). Ethanol-inducible cytochrome P4502El (CYP2El) has been linked to individual susceptibility to the hepatotoxic effect of alcohol (3). However, the results of previous studies associating genetic polymorphism at the CYP2El locus and susceptibility to ALD are contradictory (4,5). CYP2El represents the key enzyme in the non-ADH oxidation of ethanol producing acetaldehyde, an established cytotoxin (6). CYP2El-mediated metabolism often generates more toxic (reactive free ATTY
Received 1 April; revised I7 June; accepted 14 July 1997
Correspondence: Ulrich Klotz, Dr. Margarete FischerBosch-Institut ftir Klinische Pharmakologie, Auerbachstr. 112, D-70376 Stuttgart, Germany. Tel: 071 l-81013702. Fax: 071 l-859295.
Key words: Alcoholic liver disease; Chlorzoxazone; Cytochrome P450.
1009
et al.
K. Dilger
with progressingly liver disease.
severe manifestations
duced; four women and 16 men), 14 patients with more pronounced ALD (three induced, eight non-induced, three tested twice induced and later non-induced; six women and eight men) and 22 patients with severe ALD (two induced, 20 non-induced; three women and 19 men). The diagnosis of ‘severe ALD’ (alcoholic liver cirrhosis) was based on clinical signs and symptoms (esophageal varices, ascites, splenomegaly), ultrasonic evidence of cirrhosis and deranged liver function tests (albumin, prothrombin time, bilirubin). Each patient with severe ALD was graded according to the Child classification (17) into class A (n=2), class B (n= 12) or class C (n=8). ‘More pronounced ALD’ (probably alcoholic hepatitis) was diagnosed if there were no clinical or ultrasonic signs of liver cirrhosis but elevated serum alanine aminotransferase and bilirubin levels (AST>SO U/l and total bilirubin > 1.4 mgidl). In seven patients, the diagnosis of alcoholic hepatitis was confirmed by liver biopsy. Alcohol abusers without clinical or ultrasonic evidence of liver cirrhosis, but with typical ultrasonic findings of fatty liver and AST and total bilirubin below the above limits, were assigned to the group of patients with ‘minor ALD’ (probably only fatty liver). Fourteen age-matched healthy Caucasians (five women and nine men) participated as controls. Health status was examined on the basis of history, physical examination and laboratory findings. They represented teetotallers (serum ethanol not detectable) and they did not take any medications. None was obese. Two subjects were smokers. Clinical and laboratory data of patients and healthy controls are given in Table 1.
of alcoholic
Materials and Methods Subjects All patients and control subjects were tested for chlorzoxazone metabolism, after informed consent was obtained from each individual, using a protocol that was approved by the ethics committee of our hospital according to the ethical guidelines of the 1975 Declaration of Helsinki. Fifty-six Caucasian inpatients with the diagnosis of alcoholic liver disease were included in the study. Alcohol consumption was evaluated by a face-to-face-interview concerning daily ethanol consumption, duration of alcohol abuse and time of last drink. Men (women) who had been drinking more than 60 g (20 g) of ethanol daily for at least 5 years were defined as ‘alcohol abusers’. Alcohol abusers were considered as ‘induced’ if their last drink was within the 48 h preceding the chlorzoxazone intake. Alcohol abusers were considered as ‘non-induced’ after at least 6 days of alcohol withdrawal, with serum ethanol concentrations below 0.1 g/l (limit of detection of TDx/ TDxFLx REA@ ethanol assay, Abbott, Wiesbaden, Germany). Patients with pancreatitis, HIV-infection, a positive hepatitis B surface antigen or a positive antiHCV (second-generation antibody) test were excluded from the study. Patients did not receive any medication that might interfere with CYP2El activity (paracetamol, disulfiram, isoniazid, chloramphenicol, halogenated inhalational anesthetics, clomethiazole). Diabetes, serious obesity and smoking might alter CYP2El activity and were therefore recorded: four patients were non-insulin-dependent diabetics, four had a body mass index greater than 30 kg/m2 and 27 were smokers. Blood samples were obtained from 20 alcohol abusers with minor ALD (three induced, 13 non-induced, four tested twice induced and later non-in-
TABLE
Chlovzoxazone test Early in the morning after a 12-h fast, each subject received a 500 mg tablet of chlorzoxazone (Parafon forte DSC@, McNeil Pharmaceutical, Spring House, NJ, USA) orally with sips of water. A venous blood
1
Clinical and laboratory data severe alcoholic liver disease
(normal
ranges
in parentheses)
Healthy n=14 Consumption
of ethanol,
Age, yr BMI, kg/m’ (obesity >30) AST, U/l (2-19) GGT, U/l (d 6-18, P 4-18) Albumin, g/d1 (3.5-5) Bilirubin, mgidl (0.2-l .4) PT, % (70-100)
controls
g/day 58.92 11.8 25.422.9 10.92 1.6 13.425.7 4.220.3 0.7-co.3 99.122.3
of healthy
controls
Minor n=17
ALD
131-c89 51.9t11.1 23.324.0 24.52 12.2** 164?210** 3.920.8 l.OiO.8 94.429.7
and non-induced
patients
More pronounced n=ll
ALD
148~ 124 49.528.5 25.123.2 107-t52*** 1345t1474*** 3.820.4 3.7t5.7** 89.5% 13.9
Data are means?SD. Abbreviations: ALD, alcoholic liver disease; BMI, body mass index; AST, alanine transferase; PT. prothrombin time; significances (patient groups vs. healthy): **p
1010
with minor,
more pronounced
and
Severe ALD n=20 111286 59.627.6 25.2~4.2 32t28*** 200-+353*** 3.1 t0.6*** 3.7-+4.5*** 57.8%10.3***
aminotransferase;
GGT.
y-glutamyl-
CYP2El and alcoholic liver disease
sample was withdrawn 2 h after drug intake to measure chlorzoxazone and its major metabolite 6-hydroxychlorzoxazone. Thereafter fasting was stopped. Serum was stored at -20°C until analysis. Analytical
methods
Chlorzoxazone and 6-hydroxy-chlorzoxazone were determined by HPLC as described previously (18). In brief, 0.5 ml of serum was added to 0.5 ml of 0.1 M sodium acetate buffer (pH 4.0) and 10 ,ul internal standard solution (pentoxifylline 1 mg/ml) and hydrolysed overnight at 37°C with 30 ,nl of P-glucuronidase solution from Helix pomatia @-Glucuronidase/Arylsulfatase@, Boehringer Mannheim, Mannheim, Germany). Proteins were then precipitated with 0.1 ml 70% perchloric acid. Chlorzoxazone and 6-hydroxy-chlorzoxazone were extracted with 5 ml dichloromethane. The organic phases were evaporated to dryness under a stream of nitrogen. The residues were dissolved in 250 ~1 of mobile phase and 30 ,~l were injected onto a 125X4 mm RP18-column (Nucleosil 100, 5 pm, purchased from Macherey-Nagel, Dtiren, Germany). The mobile phase consisted of 8% acetonitril, 10% phosphoric acid (5%) and 82% water (v/v/v). The separated compounds were detected at 287 nm. Retention times were 8.8 min for 6-hydroxy-chlorzoxazone, 29.8 min for chlorzoxazone and 55.5 min for the internal standard. Peak height ratios were used for quantification. Calibration curves ranged from 0.5 to 20 ,@ml and from 0.5 to 4 ,&ml for chlorzoxazone and 6-hydroxychlorzoxazone, respectively. The 6-hydroxy-chlorzoxazone/chlorzoxazone concentration ratio was calculated and defined as metabolic ratio reflecting CYP2El activity Statistical
methods
The non-parametric Mann-Whitney test was used to assess the significance of differences between values for induced and non-induced alcohol abusers. By means of non-parametric ANOVA (Kruskal-Wallis, Dunn’s multiple comparisons test and post-test for linear trend, Graphpad Instat@), metabolic ratios and clinical and laboratory data were compared between three groups of non-induced patients and healthy controls. The effect of diabetes, obesity, smoking and sex on 6hydroxylation of chlorzoxazone was tested by multifactor ANOVA (Statgraphics Plus’s’). Correlation coefficients were derived by Spearman rank correlation (Graphpad Instat@).
Results No statistically significant differences between controls and patient groups were noted in age and body mass
healthy
minor
controls
ALD
more pronounced ALD
severe ALD
Fig. 1. Individual metabolic ratios in healthy controls (n= 14) and non-induced patients with minor (n=17), more pronounced (n=ll) and severe (n=20) alcoholic liver disease (ALD). Values from each group are given as means?SD. **Significantly d@erent (p
index. Consumption of ethanol (g/day) did not differ significantly between patients with minor, more pronounced and severe ALD. Metabolic ratios were measured in 15 ethanol-induced patients (seven minor, six more pronounced, two severe ALD) and 41 other noninduced patients with ALD (13 minor, eight more pronounced, 20 severe ALD). Seven (four minor, three more pronounced ALD) of the 15 induced patients were tested again after at least 6 days of ethanol withdrawal. Metabolic ratios were significantly greater in induced patients than in non-induced patients (1.1920.84, n=15 vs. 0.4320.45, n=48, mean+SD, p
olism between diabetic/non-diabetic, obese/non-obese, smoking/non-smoking and female/male subjects. In non-induced patients with severe ALD, there was no correlation between metabolic ratio and serum bilirubin (r=-0.22, p>O.O5), serum albumin (Y= -0.04, p>O.O5), prothrombin time (r=0.37, ~~0.05) and Child score (Y= -0.08, p>O.O5).
Discussion Ethanol intake induced metabolism of chlorzoxazone in alcohol abusers, Elevated CYP2El activity declined rapidly during withdrawal of alcohol. Previous studies have shown a 3-fold enhancement of the mean metabolic ratio in alcoholics tested within 2 days following ethanol withdrawal compared to alcoholics tested after 1 week of ethanol abstinence (18,19). Thus, our data are in very good agreement with these studies. Induction of CYP2El was also found in chemically-induced diabetic rats and in seriously obese humans (20-22). However, CYP2El activity was not altered in our few diabetic and obese patients. All diabetics were non-insulin-dependent and not ketoacidotic. Smoking status or sex did not appear to be of great importance when determining CYP2El activity in healthy and alcoholic subjects (18,19,23). Some investigators question the usefulness of chlorzoxazone as an in vivo probe for CYP2El activity because it has been suggested that both CYP2El and CYPlA1/2 are involved in the metabolism of chlorzoxazone. However, the contribution of CYPlA enzymes to chlorzoxazone 6-hydroxylation was considered negligible in several in vivo studies (24-28). In a recent investigation, neither human hepatic ADH nor human erythrocyte catalase contributed to chlorzoxazone metabolism, confirming the usefulness of chlorzoxazone in alcohol research on CYP2El activity (29). CYP2El activity declined continuously with severity of alcoholic liver damage in non-induced patients with ALD. We found the lowest metabolic ratios in patients with severe ALD (alcoholic liver cirrhosis). Although liver disease is well known to affect drug metabolism, the extent to which specific cytochrome P450 enzymes are impaired may vary strongly. The microsomal content of different cytochrome P450 enzymes was selectively altered in severe chronic liver disease, some being profoundly decreased, others less or not at all (14). CYP2El was reduced in microsomes of cirrhotic livers from patients with cholestasis, but not in other noncholestatic cirrhotic livers (14). In another study CYP2El levels were lower in liver samples from patients with cirrhosis than in normal samples or samples from patients with metastatic cancer (15). However, other patients with severe end-stage liver disease had 1012
normal levels of CYP2El protein (16). In viva activity of CYP2El has not yet been studied in patients with liver cirrhosis, whereas in vivo activities of several other hepatic drug metabolizing P450 enzymes, e.g. CYPlA2 and CYP2C19, were found to be reduced in these patients (30,31). It has been recommended that dosages of drugs that are substrates for disease-impaired CYPisoenzymes should be decreased in patients with liver dysfunction (9). Knowledge of altered CYP2El activity may permit better understanding or even prevention of certain adverse drug reactions, as CYP2EI is responsible for bioactivation of halothane, enflurane, isoflurane and paracetamol(32). There are consistent data indicating that both fulminant halothane hepatitis and paracetamol hepatotoxicity are associated with CYP2E 1-mediated bioactivation (33,34). Induction of CYP2E1, e.g. by ethanol, will aggravate drug-induced hepatotoxicity, whereas inhibition of CYP2EI might play a protective role. It is not known whether CYP2El is involved in fatal hepatocellular toxicity of chlorzoxazone (35). It is still an open question why some alcoholics develop severe ALD, whereas others consuming the same amount of alcohol appear to tolerate the hepatotoxic effects of ethanol. Cirrhosis morbidity starts to increase exponentially when daily alcohol consumption exceeds 60 g for male drinkers or 20 g for female drinkers (36). We studied only patients with ALD consuming ethanol beyond this critical threshold. As CYP2EI is involved in the generation of reactive free radical metabolites, oxidative species, acetaldehyde, and increased lipid peroxidation variable activity of this enzyme might explain interindividual susceptibility to ALD. Different restriction fragment length polymorphisms within the CYP2El gene have been suggested as genetic markers of susceptibility to ALD in heavy drinkers. Previous studies disagree whether alcoholics with the cl or c2 allele are at greater risk of ALD (4,5,3743). It is noteworthy that there is no relationship between genotype and phenotype of CYP2E 1, as differences in CYP2E 1 activity measured by 6-hydroxylation of chlorzoxazone in vitro and in vivo are not related to allelic forms of the CYP2El gene (25,38). Distinct phenotypes of CYP2E1, like slow and fast metabolizers. have not been described in the general population. In summary, we confirmed a 3-fold acceleration of CYP2El-mediated drug metabolism in drinking compared to abstaining patients with ALD. However, the major finding of the study was continuously decreasing CYP2El activity in abstaining patients with progressiveiy severe manifestations of ALD. It is not clear, whether alcoholic liver cirrhosis leads to reduced
CYP2El
CYP2El activity and thus impaired metabolism of chlorzoxazone or whether severe alcoholic liver injury is a consequence of altered CYP2El activity. Future investigations are necessary to determine whether CYP2El-mediated drug metabolism is reduced in all patients with advanced liver disease regardless of etiology. Nevertheless, it appears to be prudent to reduce the dosage of drugs which are substrates of CYP2El in patients with severe liver disease.
Acknowledgements 6-Hydroxy-chlorzoxazone was a kind gift of Dr. R. Boecker (University of Erlangen, Germany). This study was supported by the Robert Bosch Foundation, Stuttgart, Germany.
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